Lactoferrin-based nanoparticles as a vehicle for iron in food applications – Development and release profile
Graphical abstract
Introduction
Iron deficiency affects ca. two billion people worldwide in developing and mainly in developed countries (Martins et al., 2015). The best strategy to overcome this is to include in the diet a wide variety of foods rich in iron (Mason, Lotfi, Dalmiya, Spethuramen, & Deitchler, 2001). However, iron incorporated into complex food systems presents various problems such as oxidation and precipitation (Nicolai et al., 2011, Van der Meer et al., 1998, Wapnir, 1990). Thus, carrier systems that can actually transport and protect iron efficiently represent a field of great interest in food industry.
Several types of delivery systems at nanoscale have been developed in order to improve effectiveness and biocompatibility of bioactive compounds (Zariwala, Farnaud, Merchant, Somavarapu, & Renshaw, 2014), being nanoparticles one of these examples. These nanoparticles can be developed from natural (e.g. β-lactoglobulin and alginate) or synthetic (e.g. poly(N-isopropylacrylamide)) materials (Cerqueira et al., 2013; Fuciños et al., 2014). Additionally, they present a large surface area that can be used as a functionalization surface to specific targets, which are not accessible to macro- or microscaled particles (Cerqueira et al., 2014, Fuciños et al., 2014, Martins et al., 2015). In the food industry, the use of nanoparticles composed of proteins constitutes an interesting strategy for encapsulation and protection of micronutrients such as iron (Bourbon et al., 2015, Chen et al., 2006, Goldberg et al., 2007).
Gelling proteins, in particularly globular proteins (e.g. egg white, soy and whey proteins), have attracted much attention over the years due to their physico-chemical properties and industrial relevance (Clark et al., 2001, Nicolai and Durand, 2013). Whey proteins (such as β-lactoglobulin and lactoferrin) have been widely used in food products due to their high nutritional value and gelation capacity (Ramos et al., 2014, Xiong and Kinsella, 1990). The bovine lactoferrin (bLf) from milk is a single-chain glycoprotein of the transferrin family with 703 amino acids, folded into two globular lobules, with a molecular weight of about 80 kDa and an isoelectric point (pI) around 8–9 (Levay & Viljoen, 1995). bLf is also of interest due to its biological properties such antibacterial, antiviral, immunomodulatory and high iron binding capacity (Adlerova et al., 2008, Brock, 2002, Levay and Viljoen, 1995). In order to form gels, bLf requires thermal treatment or addition of an agent for protein denaturation. The temperature, pH and ionic strength, for example, affect gel characteristics (Bourbon et al., 2015, Lefèvre and Subirade, 2000, Ziegler and Foegeding, 1990). Thermal gelation of proteins usually requires a heating step to unfold the native protein, followed by an aggregation process to give a three-dimensional network (at nano-scale). Gelation of proteins is one of the most used methods for development of protein aggregates and at high concentrations is used to form gels. When low concentrations are used is possible to produce nanoparticles (Bourbon et al., 2015, Ramos et al., 2014). After the heating step where protein denatures and polymerizes, the cooling step and subsequent salt addition are the following events, which induce protein aggregation (Remondetto, Paquin, & Subirade, 2002). Some examples of commonly used salts are calcium chloride (CaCl2), chloride sodium (NaCl), magnesium chloride (MgCl2), magnesium sulfate (MgSO4) and iron (III) chloride (FeCl3) (Bourbon et al., 2016, Roff and Foegeding, 1996). In this work, a ferric salt (i.e. FeCl3) was chosen due to Fe3 + affinity to bLf which could address iron deficiency (Kanyshkova, Buneva, & Nevinsky, 2001). The gelation of proteins opens up interesting opportunities to produce innovative food-grade carriers for nutritional compounds (Remondetto et al., 2002). Therefore, bLf nanoparticles may be useful in food and pharmaceutical applications, e.g. to modify the optical or rheological properties of products, or to encapsulate and deliver bioactive ingredients. Moreover, understanding the molecules' release mechanisms by using mathematical modeling is essential for the design of nanoparticle-based delivery systems. This will allow foreseeing if the developed systems behaviour is appropriated to food products and consequently, to human consumption.
The main objectives of this study were the development and characterization of bLf-based nanoparticles as iron vehicle for food applications, and to highlight some of the factors that influence their properties. Additionally, iron release mechanisms from bLf nanoparticle at different pH were evaluated.
Section snippets
Materials
Purified bLf powder was obtained from DMV International (USA). This powder contained (expressed as a dry weigh percentage) 96% protein, 0.5% ash, 3.5% moisture and 0.012% iron (data supplied by the manufacturer). Iron chloride (III) (FeCl3) (97% purity) was obtained from Panreac (Barcelona, Spain). Phosphate buffer saline (PBS) and hydrochloric acid (HCl) (36.5–38.0% purity) were purchased from Sigma–Aldrich Chemical Co. Ltd. (St. Louis, MO, USA). Potassium chloride and nitric acid (35% purity)
Influence of pH, temperature and salt concentration in the formation of nanoparticles
The formation of protein nanoparticles is influenced by the treatment conditions applied during their production such as: protein concentration, salt source and concentration, pH and temperature. Being so, we can have different behaviours related with chemical characteristics of the protein and salt used. In this work, bLf nanoparticle formation was based on the best conditions (i.e. protein concentration, pH, temperature and holding time) reported by Bengoechea et al. (2011). However, due to
Conclusions
bLf nanoparticles with iron biding efficiency can be produced under specific conditions. The most effective conditions were the following: 0.2% bLf, pH 7, heating 75 °C for 20 min and adding 35 mM FeCl3. The developed nanoparticles exhibited high thermal, storage time and pH stability. The factor “stability” is very important for the food industry, as it determines the range of foods in that these nanoparticles could be added. The results showed that bLf nanoparticles were stable during 76 days and
Acknowledgements
Joana T. Martins, Ana I. Bourbon and Ana C. Pinheiro acknowledge the Foundation for Science and Technology (FCT) for their fellowships (SFRH/BPD/89992/2012, SFRH/BD/73178/2010 and SFRH/BPD/101181/2014). This study was supported by FCT under the scope of the strategic funding of UID/BIO/04469/2013 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684). This study was also supported by FCT under the scope of the Project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462). The authors would like to
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